1. Field of the Invention
This invention relates to the fields of confocal and non-confocal imaging of microscopic and macroscopic samples with particular emphasis on scanning beam fluorescence and photoluminescence imaging systems, including multi-photon fluorescence imaging and spectrally-resolved fluorescence imaging. Applications include imaging tissue specimens, genetic microarrays, protein arrays, tissue arrays, cells and cell populations, biochips, arrays of biomolecules, and many others. Other applications of this optical system include photodynamic therapy, image-guided microsurgery, and many others.
2. Description of the Prior Art
FIG. 1 shows one embodiment of a prior art confocal scanning laser macroscope, as described in U.S. Pat. No. 5,760,951. In this embodiment, the incoming laser beam 101 from laser 100 passes through a spatial filter and beam expander (comprised of lens 102, pinhole 104 and lens 106), and is expanded to match the diameter of the entrance pupil 112 of laser scan lens 118 (note—entrance pupil 112 as indicated on the figure simply indicates the position of the entrance pupil. A real stop if not usually placed at this position). Scanning mirrors 110 and 116 deflect the beam in a raster scan, and rotate about axes that are perpendicular to each other. These mirrors are placed close together, on either side of the entrance pupil of the laser scan lens. Laser scan lens 118 focuses the beam to a spot on the sample 120, and reflected light is collected by laser scan lens 118, descanned by scanning mirrors 116 and 110, and partially reflected by beamsplitter 108 into a confocal detection arm comprised of lens 128 and pinhole 130. A detector 132 is located behind the pinhole 130. Light reflected back from the focused spot on the sample passes through pinhole 130 and is detected, but light from any other point in the sample runs into the edges of the pinhole and is not detected. The scan mirrors are computer-controlled to raster the focused spot across the sample. A computer, represented by computer screen 134, is connected to the detector 132 to store and display a signal from detector 132. The computer provides means for displaying the signal from the detector. This confocal macroscope has properties similar to those of a confocal scanning laser microscope, except that the field of view of the microscope is much smaller.
Several other embodiments of the macroscope are presently in use. These include instruments for fluorescence and photoluminescence (including spectrally-resolved) imaging (several other contrast mechanisms are also possible), instruments in which a stage scan in one direction is combined with a beam scan in the perpendicular direction, non-confocal versions, and other embodiments. The combination of a scanning laser macroscope with a scanning laser microscope to provide an imaging system with a wide field of view and the high resolution capability of a microscope was described in U.S. Pat. No. 5,532,873.
The prior art macroscopes described herein and in the literature have some limitations. When focusing the instrument on a specimen, either to achieve best focus or for confocal slicing, focus position is achieved by changing the distance between the specimen and the laser scan lens. This is usually accomplished by raising or lowering the specimen on an adjustable or motorized specimen stage, or by raising or lowering the laser scan lens (or the macroscope itself) relative to the specimen. Some specimens are difficult to move, or too large to be placed on a specimen stage (one example is the human body, when the instrument is used for in-vivo imaging). This makes fine focus motion difficult to accomplish, and in the case of a macroscope using a liquid-immersion laser scan lens, changes the distance between the scan lens and the specimen, making it difficult to maintain a uniform layer of immersion fluid between the scan lens and specimen.